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PLoS One. 2014 Mar 20;9(3):e91859. doi: 10.1371/journal.pone.0091859. eCollection 2014.

Relaxed rotational and scrunching changes in P266L mutant of T7 RNA polymerase reduce short abortive RNAs while delaying transition into elongation.

Author information

1
Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America.
2
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
3
Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.
4
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America; Howard Hughes Medical Institutes, Urbana, Illinois, United States of America.

Abstract

Abortive cycling is a universal feature of transcription initiation catalyzed by DNA-dependent RNA polymerases (RNAP). In bacteriophage T7 RNAP, mutation of proline 266 to leucine (P266L) in the C-linker region connecting the N-terminal promoter binding domain with the C-terminal catalytic domain drastically reduces short abortive products (4-7 nt) while marginally increasing long abortives (9-11 nt). Here we have investigated the transcription initiation pathway of P266L with the goal of understanding the mechanistic basis for short and long abortive synthesis. We show that the P266L mutation does not alter the affinity for the promoter, mildly affects promoter opening, and increases the +1/+2 GTP K(d) by 2-fold. However, unlike wild-type T7 RNAP that undergoes stepwise rotation of the promoter binding domain and DNA scrunching during initial transcription, the P266L mutant does not undergo coupled rotational/scrunching movements until 7 nt RNA synthesis. The lack of rotation/scrunching correlates with greater stabilities of the initiation complexes of the P266L and decreased short abortive products. The results indicate that the increased flexibility in the C-linker due to P266L mutation enables T7 RNAP to absorb the stress from the growing RNA:DNA hybrid thereby decreasing short abortive products. Increased C-linker flexibility, however, has an adverse effect of delaying the transition into elongation by 1-2 nt, which gives rise to long abortive products. However, a mutation in the upstream promoter region greatly decreases long abortive products in P266L reactions, rendering the combination of P266L and A-15C promoter a desirable pair for efficient in vitro transcription for RNA production. We conclude that the conformational rigidity in the C-linker region conferred by the proline at position 266 is responsible for the undesirable short abortive products, but the rigidity is critical for efficient promoter clearance and transition into elongation.

PMID:
24651161
PMCID:
PMC3961267
DOI:
10.1371/journal.pone.0091859
[Indexed for MEDLINE]
Free PMC Article

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